Clinical applications of technological proteins has been limited because of their rapid clearance from the blood, due to glomerular filtration, proteolysis, hepatic uptake and immunogenicity. In recent years, chemical modifications of proteins with water-soluble polymers typified by polyethylene glycol (PEG) have been found to effectively overcome these drawbacks. For instance, PEG-modified proteins have been proven to have increased plasma half-lives and stability, and reduced immunogenicity in vivo. These effects are attributed to an increased molecular weight and steric hindrance, both of which are derived from PEG attached to proteins. However, clinical application of modified proteins has been limited yet. This is due to the conflicting effects of chemical modification of proteins ; modified proteins limited the transport from blood to tissues due to the high molecular weight, and sterically inhibited the binding to their receptor, resulting in loss of bioactivity. Nevertheless, an optimal modification-condition, which is a well-balanced tissue transport, receptor-binding, and plasma clearance, must be in existence. For the molecular design of modified proteins applicable to clinical use, the discovery of the optimal modification-condition, which is dominated by the steric hindrance and molecular weight, should be a primary concern. In this review, we indicated that the optimal modification of biological-active protein, such as TNFα, with water-soluble polymer markedly increased its bioavailability and was also reduced its toxic side-effects. This review will provide a fundamental information enabling us to design the modified proteins applicable to therapeutic use.

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Antisense DNA is promising therapeutic reagents based on specific inhibition of gene regulation. However, low membrane permeability is the major obstacle to deliver antisense DNA efficiently. To obtain the biological activity efficiently, antisense DNA should be delivered and bind to the target mRNA. Targeting efficacy of antisense DNA is critical issue from the pharmaceutical point of view. Since antisense DNA can block (1) transcription, (2) splicing, (3) maturation of mRNA, or (4) translocation, antisense DNA should be located in the nucleus. On the other hand, antisense DNA should be delivered in the cytoplasm to block the translation. Depends on the target site, antisense DNA should be located in the either cytoplasm or nucleus. To our knowledge, the factors are not still clear to alter the subcellular distribution of antisense DNA. To deliver antisense DNA to the proper target site, it is prerequisite to know transport mechanism of antisense DNA. We observed the subcellular distribution of antisense DNA under the confocal scanning microscope. And we tried to alter the subcellular distribution under the several conditions. Fluorescence labeled antisense DNA was punctately localized in the cytoplasm, little was in the nucleus. On the contrary, under the existence of lipofectin or virus, antisense DNA can be localized in the nucleus. Cytoplasmic pH was not great effect on the subcellular distribution of antisense DNA. From the results, it has feasibility to alter the subcellular distribution of antisense DNA, which could enhance the delivery efficacy.

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The vector systems described in this article have been used for inhibition of tumor growth. It will be discussed an appiications of non-viral vector systems with plasmid DNAs. The characteristics of the developed viral vector systems have been also summarized in this article. Most clinical trials employing retroviral vectors in present involve the administration of target cells transduced ex vivo. Although the majority of these studies is using retroviral vectors for gene transfer, there have been developed direct administration of vectors. One of them gene delivery by an electroporation method to transfer non-viral plasmid DNAs were compared with other transfer methods. In vivo electroporation electric pulses delivery in vivo can achieve the electropermeabilization of the cells in a tissue and allows hydrophilic substances to pass into the cell. Also, the selection of the expression promoters utilized in non-viral vector design are important things for transgene expression. We have examined the relative merits of introducing anti-oncogene ribozymes for cancers with these applications. And this method has been effectively transfered the plasmid DNAs into tumor. Thus in future our gene delivery system with theribozyme and the electroporation method as the drug will be used for cancer therapy.

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It is useful if the bioactive macromolecules, such as cytokines, could be delivered to the desired site. Liposomes have been used successfully as carriers for various agents. Targeting of liposomes to the desirable site has been also achieved to some extent, and the release of the contents at the target site has been attempted by means of thermosensitive liposomes utilizing phase transition of lipid bilayer. Thermosensitiye Iiposomes with internal solution of 2-fold higher osmotic pressure and sized through 200 nm-pore, released encapsulated macromolecules effectively when they were incubated at 40∼42°C, since transient swelling during phase transition of liposomes with hyper-osmotic internal aqueous phase causes permeation of large molecules through lipid bilayer. Long-circulating, serum stable, type of thermosensitive liposomes were also attemped to be prepared for practical usage of liposomes as a tool for macromolecule delivery.

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[in Japanese]

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For the treatment of peritoneal carcinomatoses, we have developed a new dosage format, which comprises cisplatin microcrystals suspended in oil (CDDP-Oil). The acute toxicity of intraperitoneal CDDP-Oil was studied in mice. The LD₅₀ value was 30.3 mg/kg in terms of cisplatin, which was 1.79 times that of a cisplatin aqueous solution (CDDP-Sol) of 16.9 mg/kg. There were no differences of the toxic symptoms between these two dosage forms, and any addisional side effects of cisplatin were not seen by the change of dosage form. However, the severity of body weight loss in the mice given CDDP-Oil was less than the mice given CDDP-Sol. We concluded that acute toxicity of cisplatin was redused by the change of dosage form.

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Lipiodol (LPD) is widely used as an oily contrast medium and an embolizing material for transcathether arterial embolization (TAE) therapy for hepatocellular carcinoma. Most of the anticancer drug is water-soluble such as epirubicin hydrochloride (EPI), so that it is used as an emulsion form with LPD. A w/o/w emulsion of LPD encapsulating the drug is a useful drug carrier to accumulate the water-soluble drug (EPI) in the carcinoma. We developed the LPD w/o/w emulsion encapsulating EPI by using a two-step pumping emulsification procedure. This procedure is easily carried out even in operation under sterilized condition. It was found that a large amount of the aqueous drug solution was encapsulated in the resultant w/o/w emulsion and the drug release profile from emulsion showed a sustained release pattern. The hemolysis induced by the w/o/w emulsion was much depressed compared to the EPI solution or the conventional o/w emulsion used for the TAE therapy. Therefore, the present w/o/w emulsion prepared by us is a desirable formulation for the TAE therapy and now under investigating with human subject.

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We have tried to exploit a time-controlled drug release system, a colon delivery capsule made of ethylcellulose (EC). Our colon delivery capsule is consisting of four parts, capsule body, cap, swellable substance and drug container. The cap, body and drug container were made of EC, which was water-insoluble polymer. At the bottom of the body, four micropores were made. The cap has different thickness and as adhered to the capsule body with concentrated EC solution. The drug release mechanism from the capsule was as follows. AL first, as the gastrointestinal fluid penetrates through these micropores, the swellable substance such as low substituted hydroxypropylcellulose(L-HPC) swells gradually. When the cap cannot endure the swelling pressure, the cap disintegrates and the drug in the container is released from the capsule. This release time of the drug from the capsule, namely the lag-time, is utilized for the delivery of drug to the colon. The lag-time was evaluated both in vitro and in vivo experiments using fluorescein as a model drug. In the case of an in vitro experiment, changing the cap thickness could control the release time of the drug. A good correlationship was obtained between the EC cap thickness and the release time of the drug. In the in vivo experiment, we administered these kinds of capsules having different cap thickness were administered to fasted beagle dogs. As an index of the release time of the drug from the capsule, the peak time(T_max) at which plasma fluorescein level reached to its maximum level was determined. There was a good correlationship between the mean T_max and the cap thickness. Therefore, we may state that this time-controlled release capsule will be useful for the colon delivery of drugs.

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